ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Aug. 2008, p. 2760–2766 Vol. 52, No.

8
0066-4804/08/$08.00⫹0 doi:10.1128/AAC.01674-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Antibiotic Resistance in Haemophilus influenzae Decreased, except for

␤-Lactamase-Negative Amoxicillin-Resistant Isolates, in Parallel with
Community Antibiotic Consumption in Spain from 1997 to 2007䌤
Silvia Garcı́a-Cobos,1 José Campos,1,2* Emilia Cercenado,3 Federico Román,1 Edurne Lázaro,4
Marı́a Pérez-Vázquez,1 Francisco de Abajo,4 and Jesús Oteo1
Antibiotic Laboratory, Bacteriology Service, Centro Nacional de Microbiologı́a, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain1;
Consejo Superior de Investigaciones Cientı́ficas, Madrid, Spain2; Microbiology Department, Hospital Gregorio Marañón,
Madrid, Spain3; and División de Farmacoepidemiologı́a y Farmacovigilancia, Agencia Española de
Medicamentos y Productos Sanitarios, Madrid, Spain4

Downloaded from http://aac.asm.org/ on March 20, 2019 by guest

Received 28 December 2007/Returned for modification 29 April 2008/Accepted 20 May 2008

The susceptibility to 14 antimicrobial agents and the mechanisms of aminopenicillin resistance were studied
in 197 clinical isolates of Haemophilus influenzae—109 isolated in 2007 (study group) and 88 isolated in 1997
(control group). Community antibiotic consumption trends were also examined. H. influenzae strains were
consecutively isolated from the same geographic area, mostly from respiratory specimens from children and
adults. Overall, amoxicillin resistance decreased by 8.4% (from 38.6 to 30.2%). ␤-Lactamase production
decreased by 15.6% (from 33 to 17.4%, P ⴝ 0.01), but amoxicillin resistance without ␤-lactamase production
increased by 7.1% (from 5.7 to 12.8%). All ␤-lactamase-positive isolates were TEM-1, but five different
promoter regions were identified, with Pdel being the most prevalent in both years, and Prpt being associated
with the highest amoxicillin resistance. A new promoter consisting of a double repeat of 54 bp was detected.
Community consumption of most antibiotics decreased, as did the geometric means of their MICs, but
amoxicillin-clavulanic acid and azithromycin consumption increased by ca. 60%. For amoxicillin-clavulanic
acid, a 14.2% increase in the population with an MIC of 2 to 4 ␮g/ml (P ⴝ 0.02) was observed; for azithromycin,
a 21.2% increase in the population with an MIC of 2 to 8 ␮g/ml (P ⴝ 0.0005) was observed. In both periods,
the most common gBLNAR (i.e., H. influenzae isolates with mutations in the ftsI gene as previously defined)
patterns were IIc and IIb. Community consumption of trimethoprim-sulfamethoxazole decreased by 54%, while
resistance decreased from 50 to 34.9% (P ⴝ 0.04). Antibiotic resistance in H. influenzae decreased in Spain from
1997 to 2007, but surveillance should be maintained since new forms of resistances may be developing.

Haemophilus influenzae is an important human pathogen
that causes community-acquired respiratory tract and invasive
infections. The increasing prevalence of antibiotic resistant
phenotypes reported in this pathogen limits the choice of a
suitable antimicrobial treatment. Antibiotic surveillance stud-
ies are necessary to determine trends in national, regional, and
local susceptibility patterns and to effectively guide empirical
antimicrobial therapy.
The principal resistance mechanism to aminopenicillins de-
scribed in H. influenzae is ␤-lactamase production, mainly of
the TEM-1 type and sometimes of the ROB-1 type (16). The
TEM ␤-lactamase gene can be associated with different pro-
moter regions: P3, overlapping Pa/Pb, Pdel, which has a 135-bp
deletion, and the newly described Prpt that includes a 54-bp
insertion in the promoter region (20, 24, 25). These promoters
may have different affinities for RNA polymerase, altering
␤-lactamase expression and giving rise to different levels of
resistance to ␤-lactam antibiotics (25).
The use of oral cephalosporins and amoxicillin-clavulanate
has contributed to the spread of ␤-lactam resistance due to
* Corresponding author. Mailing address: Centro Nacional de Mi-
crobiologı́a, Instituto de Salud Carlos III, Carretera Pozuelo a Maja-
dahonda, 28220 Majadahonda, Madrid, Spain. Phone: (34) 91-822-
3650. Fax: (34) 91-509-7966. E-mail: jcampos@isciii.es.
䌤
Published ahead of print on 27 May 2008.
amino acid substitutions in penicillin-binding proteins (PBPs),
resulting in ␤-lactamase-non-producing ampicillin resistance
(BLNAR) (7, 11, 27). In addition, the combination of ␤-lacta-
mase production and alterations in the PBP3 has given rise to
a ␤-lactamase-producing amoxicillin-clavulanic acid-resistant
(BLPACR) phenotype (13, 19, 26).
Over the years, the number of antibiotics available for treat-
ing respiratory infections has changed considerably. For in-
stance, the use of trimethoprim-sulfamethoxazole, tetracy-
clines, and amoxicillin has declined in some countries, while
the use of new macrolides, quinolones, and amoxicillin-clavu-
lanic acid has increased. However, the impact of these impor-
tant changes on the antibiotic susceptibility of respiratory H.
influenzae has received little attention.
In Spain, high levels of antibiotic resistance have been re-
ported, particularly in capsulated isolates of H. influenzae types
b, e, and f (1, 3, 4). However, widespread vaccination in chil-
dren against H. influenzae type b was established in this country
in 1998 and, in recent years, the health authorities have imple-
mented nationwide campaigns for the prudent use of antibiot-
ics, especially in children.
Accordingly, the aims of the present study were (i) to deter-
mine the current antimicrobial susceptibility of clinical isolates
of H. influenzae in Spain compared to a control group of
isolates collected 10 years ago, before the vaccination against
H. influenzae type b was introduced; (ii) to compare trends of

2760
VOL. 52, 2008 DECREASING H. INFLUENZAE RESISTANCE 2761

antibiotic susceptibility in H. influenzae in relation to the evo- TABLE 1. Comparison between the H. influenzae study group
lution of antibiotic consumption by the community; and (iii) to isolated in 2007 and the control group isolated in 1997
study the prevalence and evolution of the most important No. of isolates (%) for: Fisher exact test
mechanisms of resistance to amoxicillin, paying special atten- Parameter
2007 (n ⫽ 109) 1997 (n ⫽ 88) OR (95% CI) P
tion to modifications in PBP3 and ␤-lactamase genes and their
promoter regions. Age
Adults 35 (32.1) 32 (36.4) 0.83 (0.46–1.50) 0.55
Children 61 (56) 49 (55.7) 1.01 (0.57–1.78) 1.00
Unknown 13 (11.9) 7 (7.9) 1.57 (0.60–4.11) 0.48
MATERIALS AND METHODS
Bacterial isolates. The antibiotic susceptibility of 197 clinical isolates of H. Clinical source
influenzae was studied: 109 of them were isolated in 2007 and constituted the Respiratory 102 (93.6) 76 (86.4) 2.30 (0.86–6.12) 0.10
Conjunctivitis 43 (39.4) 37 (42) 0.90 (0.50–1.59) 0.77
study group, while 88, isolated in 1997, made up the control group. All were
Otitis 16 (14.7) 9 (10.2) 1.51 (0.63–3.60) 0.39
consecutively isolated from January to March from the same geographic area Sputum 5 (4.6) 3 (3.4) 1.36 (0.31–5.86) 0.73
covered by the university Hospital Gregorio Marañón, Madrid, Spain. This Other 37 (34) 24 (27.3) 1.37 (0.74–2.53) 0.35
hospital was chosen for the present study because it is one of the largest hospitals respiratorya
in Spain (1,700 beds), covering a large administrative health area (approximately Invasive 2 (1.8) 5 (5.7) 0.31 (0.06–1.60) 0.25

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756,000 inhabitants), and it systematically submits all its H. influenzae clinical Other 5 (4.6) 7 (7.9) 0.56 (0.17–1.82) 0.38
isolates to our Haemophilus Reference lab located 20 km away. All isolates came
Microbiological data
from individual patients, and no outbreaks or nosocomial infections due to H. Biotype I 13 (11.9) 13 (14.8) 0.78 (0.34–1.79) 0.67
influenzae were detected either in 2007 or in 1997. We have previously docu- Biotype II 53 (48.6) 45 (51.1) 0.90 (0.51–1.59) 0.77
mented that in the Madrid area nontypeable H. influenzae strains, as in other Biotype III 32 (29.3) 23 (26.1) 1.17 (0.62–2.20) 0.63
studies, are largely genetically unrelated (2, 10, 13). Biotypes IV, V, 11 (10.1) 7 (7.9) 1.30 (0.48–3.50) 0.80
Isolates were identified according to standard microbiological methods (5) and and VI
Capsulated strains 1, type f (0.9) 2, type b (2.3) 0.40 (0.04–4.50) 0.59
were serotyped by a coagglutination test (Phadebact Haemophilus test; Boule
Diagnostics AB, Huddinge, Sweden) that was confirmed by a molecular capsular a
This value includes bronchial aspirate, bronchoalveolar lavage, and respira-
typing method (8). tory secretion samples.
Antimicrobial susceptibility testing. The antibiotic susceptibility of all H. in-
fluenzae clinical isolates was determined at the same time by the broth microdi-
lution method according to the CLSI guidelines (6) on Haemophilus test me-
dium. Microtiter plates (Sensititre Enzyme 13; Trek Diagnostics, Inc., Westlake, The purified products were sequenced by using an Applied Biosystems BigDye
OH) were inoculated to produce a final density of approximately 5 ⫻ 105 Terminator v3.1 cycle sequencing kit (Perkin-Elmer, Warrington, United King-
CFU/ml, which was regularly controlled by colony counting on chocolate agar dom) according to the instructions of the manufacturer. The products were
after overnight incubation. The inoculated plates were incubated at 35°C for 20 resolved and analyzed with an ABI Prism 377 DNA sequencer. Nucleotide
to 24 h in 5% CO2. The MIC was defined as the lowest concentration of sequences were analyzed with DNAstar (Madison, WI) software; the Rd (ATCC
antibiotic that inhibited growth. Amoxicillin, amoxicillin-clavulanic acid (2:1 ra- 51907) strain (9) was included in sequence alignments as a reference.
tio), cefaclor, cefuroxime, cefotaxime, cefixime, cefpodoxime, chloramphenicol, Genotype definition. We classified our H. influenzae strains into four main
tetracycline, ciprofloxacin, nalidixic acid, telithromycin, clarithromycin, azithro- genotypes: gBLNAS, strains without a detectable resistance mechanism;
mycin, and trimethoprim-sulfamethoxazole were evaluated. gBLNAR, strains with mutations in the ftsI gene belonging to previously defined
␤-Lactamase production was determined by the chromogenic cephalosporin gBLNAR groups (7, 27); gBLPAR, strains producing ␤-lactamase and without
test with nitrocephin as substrate (21). ftsI amino acid substitutions; and gBLPACR, strains that both produce ␤-lacta-
Antibiotic quality control results for H. influenzae ATCC 49247 and ATCC mase and have amino acid substitutions in the ftsI gene (15, 18).
49766 were within recommended values according to guidelines for MIC tests Statistical analyses. Differences in the prevalence of antibiotic resistance were
(6). assessed by the Fisher exact test. The MIC values data based on double dilution
␤-Lactamase-negative isolates, which were nonsusceptible to amoxicillin (MIC of the antibiotics were converted into log2 values; the log2 values were compared
of ⱖ2 ␮g/ml), were categorized as intermediate (amoxicillin MIC of 2 ␮g/ml) and by using the unpaired t test. Associations were determined by calculation of odds
resistant (amoxicillin MIC of ⱖ4 ␮g/ml), as previously described (10). ratios (OR) with 95% confidence intervals (CI). A P value of ⬍0.05 was con-
PCR and DNA sequence determinations. Amplification of the ftsI gene be- sidered statistically significant. Statistical analyses were performed by using the
tween nucleotide positions bp 936 and 1640, corresponding to amino acid posi- GraphPad Prism version 3.02 (GraphPad Prism Software, Inc.) computer pro-
tions 327 to 540, was carried out using the primers and PCR conditions described gram.
previously (7). Antibiotic consumption. Antibiotic consumption in Spain was determined
The blaTEM gene and its promoter region were assessed by PCR using the according to the ATC/defined daily dose(s) (DDD) methodology as previously
following olignucleotide primer pairs (20): forward (5⬘-AAT TCT TGA AGA described (11, 28) and expressed as DDD per 1,000 inhabitants per day.
CGA AAG GG-3⬘) and reverse (5⬘-GTG TTA TCA CAC ATG GTT ATG-3⬘) Nucleotide sequence accession number. The nucleotide sequence data of the
(from nucleotides 3740 to 4341 of the Ampr gene transposed on plasmid 2Prpt promoter reported here is listed in the GenBank nucleotide database
pBR322, GenBank accession no. V00613) and forward (5⬘-CAT AAC CAT under accession no. EU531509.
GAG TGA TAA CAC-3⬘) and reverse (5⬘-ACG CTC AGT GGA ACG AAA
AC-3⬘) (from nucleotides 4321 to 4949). Sequencing was performed in forward
and reverse directions, and the sequences were compared to the nucleotide RESULTS
sequence of the blaTEM-1 gene and its promoter region in public databases
(GenBank accession no. AB194682). Clinical isolates. The clinical and microbiological features
The PCR program for ftsI and tem genes was as follows: denaturation at 94°C of the collected isolates from 2007 and 1997 were similar
for 5 min, 25 amplification cycles of denaturation at 94°C for 30 s, annealing of (Table 1).
primers at 55°C for 30 s, and primer extension at 72°C for 30 s were carried out,
Antibiotic susceptibility. (i) Overall susceptibility. The an-
followed by a final primer extension step at 72°C for 7 min.
Reactions were performed in a 25-␮l final volume, with the PureTaq Ready- tibiotic MIC90s for H. influenzae in 2007 and 1997 were similar,
To-Go PCR bead method (Amersham Biosciences), containing 5 ␮l of DNA although some differences were observed in the geometric
template, 10 mM Tris-HCl (pH 9), 50 mM KCl, 1.5 mM MgCl2, 1 ␮M concen- means (Table 2). Statistical comparisons of the MICs using the
trations of each primer, 200 ␮M concentrations of each deoxynucleoside triphos- unpaired t test revealed significant differences between the two
phate, and 2.5 U of Taq polymerase. PCR products were visualized by 1%
agarose gel electrophoresis and ethidium bromide staining, and all PCR products
populations for five antibiotics: amoxicillin (P ⫽ 0.05), cefaclor
were purified with a PCR purification kit (Qiagen, Inc.) and were subjected to (P ⫽ 0.003), chloramphenicol (P ⫽ 0.008), azithromycin (P ⫽
automated DNA sequencing. 0.0001), and trimethoprim-sulfamethoxazole (P ⫽ 0.03) (Table
2762 GARCÍA-COBOS ET AL. ANTIMICROB. AGENTS CHEMOTHER.

TABLE 2. Antibiotic susceptibility distributions of 14 antimicrobials against 197 isolates of H. influenzaea

MIC (␮g/ml)
Geometric mean
Antibiotic MIC50 MIC90 Range Pb

1997 2007 1997 2007 1997 2007 1997 2007 % Variation

Amoxicillin 1 0.5 ⱖ64 32 ⱕ0.25–ⱖ64 ⱕ0.25–ⱖ64 0.05 2.83 1.67 –41.0

Amoxicillin-clavulanic acid 0.5 0.5 2 2 0.25–4 0.25–4 0.49 0.78 0.84 ⫹7.69
Cefaclor 4 4 16 16 1–32 ⱕ0.5–32 0.003 5.23 4.43 –15.30
Cefuroxime 1 1 4 4 0.25–8 0.25–8 0.07 1.16 1.07 –7.76
Cefotaxime ⱕ0.01 ⱕ0.01 0.06 0.06 ⱕ0.01–0.12 ⱕ0.01–0.25 0.94 0.18 0.18 0.00
Cefixime ⱕ0.06 ⱕ0.06 ⱕ0.06 ⱕ0.06 ⱕ0.06–0.5 ⱕ0.06–0.25 0.66 0.063 0.062 –1.59
Cefpodoxime ⱕ0.06 ⱕ0.06 0.25 0.25 ⱕ0.06–0.25 ⱕ0.06–0.5 0.85 0.084 0.086 ⫹2.38
Chloramphenicol ⱕ0.5 ⱕ0.5 1 ⱕ0.5 ⱕ0.5–8 ⱕ0.5–8 0.008 0.63 0.54 –14.29
Tetracycline ⱕ0.5 ⱕ0.5 ⱕ0.5 ⱕ0.5 ⱕ0.5–ⱖ8 ⱕ0.5–4 0.14 0.55 0.51 –7.27
Telithromycin ⱖ2 ⱖ2 ⱖ2 ⱖ2 0.5–ⱖ2 0.5–ⱖ2 0.19 1.72 1.73 ⫹0.58

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Clarithromycin 8 8 16 16 2–32 2–16 0.65 9.15 8.91 –2.62
Azithromycin 2 2 4 4 0.25–8 0.5–8 0.0001 1.60 2.19 ⴙ36.88
Trimethoprim-Sulfamethoxazole 1 ⱕ0.25 16 16 ⱕ0.25–ⱖ64 ⱕ0.25–32 0.03 1.69 0.97 –42.60
Ciprofloxacin ⱕ0.03 ⱕ0.03 ⱕ0.03 ⱕ0.03
a
Significant values are indicated in boldface.
b
As determined by an unpaired t test.

2); no differences were observed for the remaining nine anti-
biotics. For the azithromycin MICs, the 25th percentiles were
1 ␮g/ml (for 1997) and 2 ␮g/ml (for 2007), while the 75th
percentiles were 2 ␮g/ml (for 1997) and 4 ␮g/ml (for 2007). For
amoxicillin MICs, the 75th percentiles were ⱖ64 ␮g/ml (for
1997) and 4 ␮g/ml (for 2007); for amoxicillin-clavulanic acid,
the 75th percentiles were 1 ␮g/ml (for 1997) and 2 ␮g/ml (for
2007). Most importantly, the geometric mean of azithromycin
increased 36.88% between 1997 and 2007, while it decreased
for amoxicillin (21.23%), cefaclor (15.30%), chloramphenicol
(14.29%), and trimethoprim-sulfamethoxazole (42.60%) (Ta-
ble 2). A variation in the geometric mean of ⱖ14% was sta-
tistically significant as determined by the unpaired t test.
(ii) Amoxicillin susceptibility. A total of 30.2% of the 2007
isolates had an amoxicillin MIC ⱖ 4 ␮g/ml, compared to 38.6%
in 1997. In addition, a higher proportion of ␤-lactamase posi-
tive isolates from 1997 had an amoxicillin MIC of ⱖ32 ␮g/ml
(P ⫽ 0.04) (Table 3). Nevertheless, while ␤-lactamase produc-
tion decreased by 15.5% from 1997 to 2007 (P ⫽ 0.01) (Table
3), non-␤-lactamase-mediated amoxicillin resistance increased
by 7.1% (from 5.7% in 1997 to 12.8% in 2007). In fact, the
percentage of ␤-lactamase-negative isolates with an amoxicillin
MIC of ⱖ2 ␮g/ml increased from 13.6% in 1997 to 28.9% in
2007 (P ⫽ 0.04) (Table 3).
(iii) Amoxicillin-clavulanic acid susceptibility. Resistance to
amoxicillin-clavulanic acid was not detected according to cur-
rent breakpoints (6). However, the number of ␤-lactamase-
negative isolates with an amoxicillin-clavulanic acid MIC of ⱖ2
␮g/ml was higher in 2007 (27.8%) than in 1997 (13.6%) (P ⫽
0.02) (Table 3).

TABLE 3. Antimicrobial susceptibility comparison between the H. influenzae study group isolated in 2007 and the control group isolated in 1997
No. of isolates (%) for: Fisher exact test
Parameter % Change
2007 (109) 1997 (88) OR (95% CI) Pd

Amoxicillin resistance (MIC ⱖ 4 ␮g/ml) 33 (30.2) 34 (38.6) –8.4 0.69 (0.38–1.25) 0.23
␤-Lactamase-positive isolates 19 (17.4) 29 (32.9) –15.5 0.43 (0.22–0.83) 0.01
Amoxicillin resistance in ␤-lactamase- 14 (12.8) 5 (5.7) ⫹7.1 1.99 (0.68–5.85) 0.31
negative isolates
Amoxicillin, MIC ⱖ 2 ␮g/mla 26 (28.9) 8 (13.6) ⫹15.3 2.60 (1.08–6.20) 0.04
Amoxicillin, MIC ⱖ 32 ␮g/mlb 15 (78.9) 29 (100) –21.1 2.45 (1.02–5.90) 0.04
Amoxicillin-clavulanic acid, MIC ⫽ 2 to 25 (27.8) 8 (13.6) ⫹14.2 0.05 (0.003–1.16) 0.02
4 ␮g/mlc
gBLNAS 60 (55) 43 (48.9) ⫹6.1 1.30 (0.73–2.25) 0.40
gBLPAR 16 (14.7) 17 (19.3) -4-.6 0.72 (0.34–1.52) 0.44
gBLNAR 30 (27.5) 16 (18.2) ⫹9.3 1.71 (0.86–3.40) 0.13
gBLPACR 3 (2.8) 12 (13.6) –15.4 0.13 (0.036–0.45) 0.0004
Azithromycin, MIC ⫽ 2 to 8 ␮g/ml 95 (87.2) 58 (66) ⫹21.2 3.50 (1.72–7.20) 0.0005
Trimethoprim-sulfamethoxazole, 38 (34.9) 44 (50) –15.1 0.53 (0.30–0.95) 0.04
MIC ⱖ 4 ␮g/ml
Trimethoprim-sulfamethoxazole and 16 (14.7) 23 (26.1) –11.4 0.49 (0.24–1.00) 0.04
amoxicillin simultaneous resistance
a
This values includes intermediate and resistant isolates (9). Total ␤-lactamase-negative isolates: 90 in 2007 and 59 in 1997.
b
Total ␤-lactamase-positive isolates: 19 in 2007 and 29 in 1997.
c
Total ␤-lactamase-negative isolates: 90 in 2007 and 59 in 1997.
d
Significant values are indicated in boldface.
VOL. 52, 2008
(iv) Cephalosporin susceptibility. The proportion of cefa-
clor-nonsusceptible isolates (resistant and intermediate) de-
creased 12.2% from 17% in 1997 to 4.8% in 2007. No resis-
tance to the other cephalosporins was found.
(v) Macrolide susceptibility. Clarithromycin resistance
(MIC of 32 ␮g/ml) was 0% in 2007 and 2.3% in 1997. The
proportion of isolates with an azithromycin MIC of 8 ␮g/ml
(the susceptibility breakpoint is currently defined as ⱕ4 ␮g/ml)
(6) was 0.9% in 2007 and 1.1% in 1997. However, the percent-
age of isolates with an azithromycin MIC from 2 to 8 ␮g/ml
significantly increased in isolates from 2007 in relation to iso-
lates from 1997 (P ⫽ 0.0005) (Table 3).
(vi) Trimethoprim-sulfamethoxazole susceptibility. The
prevalence of trimethoprim-sulfamethoxazole resistance de-
creased from 50% in 1997 to 34.9% in 2007, a decrease of
15.1% (P ⫽ 0.04) (Table 3). Also, resistance to trimethoprim-
sulfamethoxazole was more prevalent in amoxicillin-resistant
(57.4%) strains than in amoxicillin-susceptible strains (28.7%)
(P ⫽ 0.0002, OR ⫽ 3.34, 95% CI ⫽ 1.78 to 6.26). Simultaneous
resistance to trimethoprim-sulfamethoxazole and amoxicillin
decreased from 26.1% in 1997 to 14.7% in 2007 (P ⫽ 0.04)
(Table 3).
Amoxicillin susceptibility in relation to resistance genotype.
Of the 2007 H. influenzae isolates, 60 (55.0%) had no detect-
able mechanism of resistance (gBLNAS genotype) compared
to 43 (48.9%) in the control group from 1997 (P ⫽ 0.40).
Sixteen isolates (14.7%) were gBLPAR in 2007 compared to
17 (19.3%) in 1997; thirty isolates (27.5%) were gBLNAR in
2007 compared to sixteen isolates (18.2%) in 1997. The num-
ber of gBLPACR strains decreased from 12 isolates (13.6%) in
1997 to 3 isolates (2.8%) in 2007 (P ⫽ 0.0004) (Table 3).
The distribution of MICs is depicted in Fig. 1 according to
the mechanisms of ␤-lactam resistance. Those isolates with an
amoxicillin MIC of 2 to 4 ␮g/ml were gBLNAR, while the
highest levels of amoxicillin resistance (32 to ⱖ64 ␮g/ml) were
attained in isolates that produce ␤-lactamase, gBLPAR and
gBLPACR (Fig. 1). In the case of amoxicillin-clavulanic acid,
isolates with MICs of 2 ␮g/ml were mostly gBLNAR but also
included gBLPACR and gBLPAR, while only gBLNAR and
gBLPACR had MICs of 4 ␮g/ml (Fig. 1).
The effectiveness of ␤-lactam antibiotics against the 197 H.
influenzae isolates according to their resistance genotype
showed that the MIC90s for amoxicillin, amoxicillin-clavulanic
acid, and cefuroxime of the gBLNAR isolates were about
eightfold higher than the gBLNAS isolates, fourfold higher for
cefaclor, and threefold higher for cefotaxime (data not shown);
no differences were found between the two study periods.
Trends in the mutation patterns of the ftsI gene. Amino acid
substitutions in the ftsI gene of gBLNAR and gBLPACR iso-
lates and their susceptibility to amoxicillin are listed in Table 4.
Groups I and II had similar frequencies in H. influenzae iso-
lates from 2007 and 1997: group I, 6.1% (in 2007) and 7.1% (in
1997), and group II, 93.9% (in 2007) and 92.8% (in 1997).
However, some rearrangement was found within group II, as
group IIa increased from 0.0% (in 1997) to 15.1% (in 2007),
while groups IIb, IIc, and IId decreased by 3.8, 4.0, and 5.2%,
respectively, between 1997 and 2007.
Within group II, the most common mutations in 1997 and
2007 were groups IIb, 32.1% (1997) and 27.3% (2007), and IIc,
46.4% (1997) and 42.4% (2007). All of the ftsI mutation groups
DECREASING H. INFLUENZAE RESISTANCE 2763
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FIG. 1. Amoxicillin and amoxicillin-clavulanic acid MIC distribu-
tion for H. influenzae in relation to mechanisms of amoxicillin resis-
tance. Abbreviations: gBLNAR, H. influenzae isolates with mutations
in the ftsI gene as previously defined; gBLNAS, H. influenzae isolates
without a resistance mechanism; gBLPACR, H. influenzae isolates with
both mechanisms (␤-lactamase production and amino acid substitu-
tions in the ftsI gene); and gBLPAR, H. influenzae ␤-lactamase-pro-
ducing isolates.
had geometric means of about 2 ␮g/ml for amoxicillin (␤-
lactamase-negative isolates) and amoxicillin-clavulanic acid
(Table 4).
The miscellaneous group (group M in Table 4) had overall
prevalences of 18.2% in 1997 and 8.2% in 2007. Group M
presented geometric means of 0.5 ␮g/ml for amoxicillin and
amoxicillin-clavulanic acid (Table 4). Geometric means of 3.4,
0.84, 0.01, 0.06, and 0.06 ␮g/ml were found for cefaclor, cefur-
oxime, cefotaxime, cefixime, and cefpodoxime, respectively,
similar to those obtained in isolates without any mechanism of
resistance, suggesting that these mutations may not be associ-
ated with decreased susceptibility to these antibiotics (Table
4). Within group M, the most important mutation pattern
(eight isolates in 1997 and three isolates in 2007) was char-
acterized by the Asp350-to-Asn amino acid substitution (Ta-
ble 4).
Characterization of the promoter regions of ␤-lactamases.
Overall, the proportion of the different promoter regions
found in the ␤-lactamase-positive isolates was as follows:
39.6% (42.1% in 2007 and 37.9% in 1997) of the isolates had
the Pdel promoter, 33.3% (31.6% in 2007 and 34.5% in 1997)
had the Pa/Pb promoter, 22.9% (15.8% in 2007 and 27.6% in
1997) had the Prpt promoter, and 2.1% (5.3% in 2007 and 0%
in 1997) had the P3 promoter.
A new variant of the Prpt promoter (here designated as
2Prpt) was detected in a nontypeable clinical isolate taken in
2007 from a child with otitis media. 2Prpt consisted of a double
repeat of bp 145 to 198 inserted after bp 198 (Fig. 2). The
double insertion provides additional ⫺10 and ⫺35 sequences
2764 GARCÍA-COBOS ET AL. ANTIMICROB. AGENTS CHEMOTHER.

TABLE 4. Amino acid substitutions in the ftsI gene in H. influenzae

Amino acid substitutions Overall
Near STVK MIC AMX geometric No. of isolates
Surrounding KTG motif meanb
Group motif Thr- Lys- Leu- Met- Met- Ala- Ile- Ile- Gly- range
(␮g/ml)
Lys- Ile- Asp- 352 355 356 377 391 437 449 475 490 Ala- Arg- Ile- Asn- Ala- Thr-
AMX AMC 1997 2007 Total
344 348 350 502 517 519 526 530 532

I His Ser 1–ⱖ64 1.59 2.00 2 2 4

IIa Lys 0.5–4 2.00 1.74 3 3

Lys Ser 1–4 2 2

IIb Val Lys 2–4 2.00 2.00 2 2

Glu Val Lys 1 1 1
Asn Ile Val Lys 1–ⱖ64 4 6 10
Asn Ile Glu Val Lys 2–16 1 2 3
Asn Ser Val Leu Lys 1 2 2

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IIc Thr Lys 0.5–32 2.18 1.80 7 6 13
Asn Thr Lys 1–ⱖ64 6 8 14

IId Val Lys 0.5–ⱖ64 2.00 2.00 4 3 7

Ma Ile ⱕ0.25 0.52 0.53 1 1

Leu 0.5 2 2
Thr ⱖ64 1 1
Asn ⱕ0.25–ⱖ64 8 3 11
Asn Ser 0.5 2 2 4
Val 0.5 1 1
Val 0.5 1 1
Ser 0.5 2 2
Arg Asn Gly Thr Val Ile 0.5 1 1
Arg Asn Gly Thr Val Ile Ser ⱕ0.25 1 1
a
M, miscellaneous.
b
The amoxicillin (AMX) value was calculated for the ␤-lactamase-negative isolates only (n ⫽ 149); the amoxicillin-clavulanic acid (AMC) value was calculated for
the entire collection (n ⫽ 197).

equal to the Prpt promoter, in such a way that blaTEM-2Prpt
seems to have three promoters. The new promoter and the
promoter corresponding to Prpt are likely stronger than the
coexisting P3 promoter as the ⫺35 regions are a closer match
to the consensus sequence (Fig. 2).
The highest geometric mean for amoxicillin was observed
for isolates harboring the Prpt promoter (56.42 ␮g/ml), fol-
lowed by Pdel promoter (44.44 ␮g/ml) and Pa/Pb promoter
(41.50 ␮g/ml) (data not shown); for amoxicillin-clavulanic acid,
the geometric means were 1.13, 1.29, and 0.96 ␮g/ml for the
Prpt, Pdel, and P3 promoters, respectively (data not shown).
Antibiotic consumption. A decrease in antibiotic consump-
tion at the community level was observed for amoxicillin,
cefaclor, cefuroxime, cefixime, cotrimoxazole, and clarithromycin
(Table 5); conversely, amoxicillin plus ␤-lactamase inhibitor
and azithromycin experienced an important increase of ca.
60% (Table 5).
Simultaneous variation on antibiotic consumption and anti-
biotic susceptibility is presented in Fig. 3. For a number of
antibiotics (amoxicillin, cefaclor, cefuroxime, cefixime, cla-
rithromycin, and cotrimoxazole), both antibiotic consumption
and antimicrobial resistance decreased over the study period.
In contrast, amoxicillin-clavulanic acid and azithromycin expe-
rienced parallel increases in antibiotic consumption and anti-
biotic geometric means (Fig. 3).
DISCUSSION
Several observations of clinical and epidemiological interest
can be made from the data presented here. First, the propor-

FIG. 2. Promoter region (bp 1 to 208) of blaTEM (23). Underlining represents region in P3 which is replicated once in Prpt and twice in 2Prpt.
*, Point of insertion in P3 to create 2Prpt (text in boldface represents inserted sequences). Boxed regions represent ⫺10 and ⫺35 sequence of
promoter P3, circled regions represent ⫺10 and ⫺35 sequence of promoter Prpt, and dotted circles represent new ⫺10 and ⫺35 regions in 2Prpt.
VOL. 52, 2008 DECREASING H. INFLUENZAE RESISTANCE 2765

TABLE 5. Community antimicrobial consumption in Spain over long periods of time altering the antibiotic consumption
Consumption by the community has important consequences on the antibi-
(DDD/1,000 otic susceptibility of H. influenzae. A decrease in antibiotic
inhabitants/ Variation
WHO ATC code Antibiotic
(%)
resistance is only occasionally observed in the scientific litera-
day) in:
ture, but it is well documented here for amoxicillin (a 36.9%
1997 2006 decrease in consumption, a 8.4% decrease in resistance, and a
J01C ␤-Lactam antibacterials, 15.5% decrease in ␤-lactamase production) and trimethoprim-
penicillins sulfamethoxazole (a 54.1% decrease in consumption and a
J01CA04 Amoxicillin 6.5 4.1 –36.9 15.1% decrease in resistance). In addition, simultaneous resis-
J01CR02 Amoxicillin plus ␤-lactamase 4.4 7.2 ⫹63.6
inhibitor tance to amoxicillin and trimethoprim-sulfamethoxazole de-
creased by 11.4%.
J01D Cephalosporins Furthermore, in addition to the decreasing prevalence of
J01DC04 Cefaclor 0.4 0.1 –75.0 ␤-lactamase, the H. influenzae population with an amoxicillin
J01DC02 Cefuroxime 1.2 1.0 –16.7
MIC of ⱖ32 ␮g/ml decreased. These results are in accordance

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J01DD08 Cefixime 0.5 0.3 -40.0
with data from a recent surveillance study in the United States
J01EE01 Trimethoprim- 0.7 0.3 –54.1 and with European studies, which confirmed a decrease in the
sulfamethoxazole incidence of ␤-lactamase production in this pathogen (14, 17).
J01FA Macrolides Two antibiotics, amoxicillin-clavulanic acid and azithromy-
J01FA09 Clarithromycin 1.2 0.9 –25.0 cin, demonstrated an increase in consumption over the study
J01FA10 Azithromycin 0.5 0.8 ⫹60.0 period of ca. 60%. The increases were recent, and we noticed
little impact on true antibiotic resistance as measured by cur-
rent breakpoints. However, we have documented some
tion of H. influenzae isolates producing ␤-lactamase decreased changes in the H. influenzae population that may indicate on-
by 15.5% between two sets of isolates collected 10 years apart. going adaptations to this increase in antibiotic pressure. For
Second, because the proportion of resistant isolates without amoxicillin-clavulanic acid, we observed a 14.2% increase in
␤-lactamase production increased by 7.1%, overall amoxicillin the H. influenzae population presenting an MIC of 2 to 4 ␮g/ml
resistance decreased by only 8.4% (from 38.6 to 30.2%). Fi- (P ⫽ 0.02), and for azithromycin we found a 21.2% increase in
nally, while all ␤-lactamase-positive isolates were of the clas- the population presenting an MIC of 2 to 8 ␮g/ml (P ⫽ 0.0005).
sical TEM 1 type, up to five different promoter regions were In our study, the percentage of isolates with an amoxicillin
detected. The Pdel promoter was the most prevalent in both MIC of ⱖ2 ␮g/ml (intermediate and resistant) (10) without
years, followed by the Prpt promoter, but the prevalence of ␤-lactamase production increased from 13.6% in 1997 to
Prpt decreased by 11.8% from 1997 to 2007, while the propor- 28.9% in 2007, while the gBLNAR group increased from
tion of Pdel increased by 4.2%; these changes may be impor- 18.2% in 1997 to 27.5% in 2007. However, in Europe and the
tant because the Prpt promoter showed the greatest resistance United States the number of ampicillin-resistant non-␤-lacta-
to amoxicillin (data not shown). mase-producing strains remained relatively constant and low in
One possible limitation of the present study was that sus- recent years (14, 17). This difference may be the consequence
ceptibility data from one hospital were compared to national of the large amoxicillin-clavulanic acid consumption detected
average consumption data. The data from Table 2 suggest that in Spain that is among the highest in Europe (12). In accor-

FIG. 3. Comparison of percentages of variation between community antibiotic consumption and antibiotic activity in H. influenzae (Spain, 1997
to 2007). Drug abbreviations: AMX, amoxicillin; AMC, amoxicillin-clavulanic acid; CEC, cefaclor; CXM, cefuroxime; CFM, cefixime; CLR,
clarithromycin; AZM, azithromycin; and SXT, trimethoprim-sulfamethoxazole.
2766 GARCÍA-COBOS ET AL.
dance with data previously reported by us (11), the vast ma-
jority of the gBLNAR isolates in the present study had amino
acid substitutions at the Lys-Thr-Gly motif, the most common
being Asn-526 to Lys. However, in the present study no amino
acid substitutions were found at positions 385 and 389, previ-
ously found to be related to a decreased susceptibility to cefo-
taxime and cefixime (11, 22).
As in other reports (20, 24), in our ␤-lactamase-positive
isolates the most prevalent promoter was Pdel. The geometric
mean for amoxicillin was higher for the Pdel promoter than for
the Pa/Pb promoter, as reported by Tristram et al. (24). In our
study, the Prpt promoter presented the highest geometric
means for amoxicillin and cefaclor, suggesting that it may be
associated with increased levels of resistance to these two an-
tibiotics, but a number of additional factors such as plasmid
location and plasmid copy numbers may also determine the
level of susceptibility.
In summary, we report here data that are of clinical and
epidemiological interest that may be important to other coun-
tries as well. Overall, H. influenzae antibiotic resistance de-
creased in Spain from 1997 to 2007. In this period, nationwide
campaigns for the prudent use of antibiotics have been imple-
mented by the health authorities and conjugate vaccines
against H. influenzae type b (a microorganism that was highly
antibiotic resistant in Spain and constituted a reservoir for
antibiotic resistance genes) were introduced. However, antibi-
otic surveillance studies on this pathogen should be maintained
since we noticed that amoxicillin-clavulanic acid consumption,
as well as amoxicillin resistance, in ␤-lactamase negative iso-
lates experienced a relevant increase. Also, the azithromycin
MICs in the study population increased in parallel with the
important increase observed in the community consumption of
azithromycin.
ACKNOWLEDGMENTS
This study was supported by research grants from the Instituto de
Salud Carlos III, Fondo de Investigaciones Sanitarias (reference 04/
0899), the REIPI Network (reference RD 06/0008/0023), and the Net-
work of Excellence GRACE (PL 518226). S.G.-C. is a recipient of a
fellowship from the Instituto de Salud Carlos III (reference 05/0033).
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